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Mol Biol Cell
Mol Biol Cell
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Molecular Biology of the Cell
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Articles
Nkd1 functions downstream of Axin2 to attenuate Wnt signaling
Bell Ian a
Khan Haider a
Stutt Nathan b
Horn Matthew a
Hydzik Teesha a
Lum Whitney a
Rea Victoria a
Clapham Emma a
Hoeg Lisa c
Van Raay Terence J. a *
a Department of Molecular and Cellular Biology, University of Guelph, Guelph, N1G 2W1 Ontario, Canada
b Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
c Department of Bioinformatics, University of Guelph, Guelph, Ontario, N1G 2W1 Canada
Wallingford John Monitoring Editor
University of Texas, Austin
Authors contributions: I.B. and T.V.R. conceived and planned the experiments. H.K. and N.S. made the nkd1gh/gh2 mutant. M.H., E.C., and I.B. conducted the BIO experiments. H.K. and I.B. carried out the qRT-PCR analysis. T.H. and I.B. carried out the western blotting analysis. V.R. and L.H. carried out the DTU analysis. W.L. performed Axin2 WMISH and gsc arc angle with Axin2 OE and Axin2 MO knockdown. All other experiments were carried out by I.B. I.B. and T.V.R. wrote the manuscript. M.H., I.B., and T.V.R. edited the manuscript.

*Address correspondence to: Terence J. Van Raay (tvanraay@uoguelph.ca)
01 7 2024
01 7 2024
35 7 ar9306 2 2024
10 4 2024
19 4 2024
© 2024 Bell et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 4.0 Unported Creative Commons License.

Wnt signaling is a crucial developmental pathway involved in early development as well as stem-cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic β-catenin, and Nkd1 functions to inhibit the nuclear localization of β-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2gh1/gh1, nkd1gh2/gh2 single mutants and axin2gh1/gh1;nkd1gh2/gh2 double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2gh1/gh1;nkd1gh2/gh2 double mutants. Further, Wnt target gene expression by qRT-PCR and RNA-seq, and protein expression by mass spectrometry demonstrated that the double axin2gh1/gh1;nkd1gh2/gh2 mutant resembled the nkd1gh2/gh2 phenotype demonstrating that Nkd1 functions downstream of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of β-catenin-dependent transcription having novel readouts of Wnt activity compared with nkd1gh2/gh2 or the axin2gh1/gh1;nkd1gh2/gh2 double mutant. We also investigated the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/β-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.

Regulation of Wnt signaling is critical in development and in the maintenance of stem cells, but how this regulation is controlled by feedback regulators is poorly understood.

Here, we knock out two constitutive negative feedback regulators that traditionally have been considered as independent regulators of Wnt signaling and found that Nkd1 functions downstream of Axin2 in the pathway to inhibit β-catenin dependent transcriptional activity.

These results allow us to envisage a system where we can manipulate the negative feedback mechanisms to control the levels of Wnt signaling under conditions of exacerbated wnt signaling.
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pmcINTRODUCTION

Canonical Wnt signaling is important in regulating many events during early development (Kim et al., 2000; Kosinski et al., 2007; Lu et al., 2011; Russell and Monga, 2018; Wang et al., 2018). Dysregulation of Wnt signaling, primarily due to mutations causing the pathway to become hyperactive, have been linked to many diseases including cancer, diabetes, bone density defects, and neurological disorders (Gong et al., 2001; Novellasdemunt et al., 2015; Xu et al., 2020; Chen et al., 2021; Gao et al., 2021; Yu et al., 2022). Wnt signaling has been described extensively elsewhere (Komiya and Habas, 2008; Zeng et al., 2008; Flack et al., 2017), but briefly, when Wnt signaling is turned on, cytoplasmic levels of β-catenin stabilize, allowing for its translocalization to the nucleus where it interacts with various coactivators to transcriptionally activate Wnt target genes (Borrelli et al., 2021). Two common Wnt target genes are axin2 and nkd1, which function as negative feedback regulators for the pathway (Angonin and Van Raay, 2013; Bernkopf et al., 2015).

Nkd1 is an antagonist in both the canonical and noncanonical Wnt signaling pathways (Van Raay et al., 2007; Marsden et al., 2018; Wang et al., 2021b). At the N-terminal end of Nkd1 is a myristoylation sequence which acts as a reversible membrane localization switch that is necessary for its activity in both canonical and noncanonical Wnt signaling (Hu et al., 2010; Van Raay et al., 2011). Nkd1 contains several other conserved domains, including a poly histidine tail that has been shown to interact with Axin and Axin2 (Wharton et al., 2001; Rousset et al., 2002; Miller et al., 2009; Gammons et al., 2020). The model put forth by Gammons et al suggests that the poly-histidine tail is required for Nkd1’s aggregation that is dependent on its interaction with Dvl. The model suggests that aggregation of Nkd1 ensures that it only functions after sustained Wnt signaling (Gammons et al., 2020). Further, we demonstrated that Nkd1’s function and interaction with β-catenin is Wnt ligand dependent, functioning to inhibit β-catenin nuclear localization only in the presence of a Wnt ligand (Van Raay et al., 2011; Larraguibel et al., 2015).

Loss of Nkd1 in Drosophila results in a naked cuticle phenotype in the larva and in wing patterning defects later in development, both of which demonstrate that Nkd functions to inhibit canonical Wnt signaling (Zeng et al., 2000; Wang et al., 2021a). In mice, loss of Nkd1 and/or Nkd2 has subtle defects in cranial bone morphology, slightly reduced litter sizes and effects on spermatogenesis, but are otherwise viable (Li et al., 2005; Zhang et al., 2007). In zebrafish, we demonstrated that knockdown of Nkd1 results in exacerbated and sensitized Wnt signaling (Van Raay et al., 2007). Finally, misexpression of Nkd’s in multiple systems further demonstrates that Nkd’s act as Wnt signaling antagonists (Wharton et al., 2001; Van Raay et al., 2007, 2011; Schneider et al., 2010; Larraguibel et al., 2015; Marsden et al., 2018).

Axin1 and Axin2 serve similar functions, acting as scaffolding proteins in the assembly of the destruction complex. They have unique expression patterns with axin1 being more ubiquitously expressed whereas axin2 expression is more restricted to cells undergoing active Wnt signaling (Zeng et al., 1997; Yamamoto et al., 1998; Lange et al., 2023). Furthermore, Axin1 appears to be a more potent inhibitor of canonical Wnt signaling as overexpression of Axin1 reduces β-catenin protein levels more than overexpression of Axin2 as seen by immunocytochemistry in SW480 cells (Bernkopf et al., 2015). However, when Wnt signaling is activated, Axin2 is a more potent antagonist for the pathway as knocking down Axin2, but not Axin1, increased dephosphorylated levels of β-catenin in the presence of the Wnt ligand revealing that Axin1 cannot always compensate for the loss of Axin2 (Bernkopf et al., 2015). Furthermore, both Axin1 and Axin2 have unique domains that affect their ability to self-polymerize (Fiedler et al., 2011; Bernkopf et al., 2019) and one study found that Axin1, but not Axin2, binds to Dvls (Miller et al., 2009). The differential importance for Axin1 and Axin2 during development is highlighted in knockout mouse models as loss of Axin1 is lethal whereas loss of Axin2 has no obvious phenotype during early embryonic development (Yu et al., 2005; Xie et al., 2011). Later in development, Axin2 knockout mice have a small head phenotype, delayed heart valve maturation, and thickened aortic valve but are viable and fertile (Yu et al., 2005; Hulin et al., 2017).

In vitro, it has been shown that Nkd1 interacts with both Axin1 and Axin2 (Miller et al., 2009). Curiously, Axin1 only interacted with a high molecular weight aggregate form of Nkd1 with the interaction being abolished by mutating the C-terminal histidine cluster on Nkd1 (Gammons et al., 2020). Nkd1 interaction with Axin1 could be important for its function as knocking down Axin1 in HEK293T cells reduced Nkd1’s ability to antagonize Wnt3a activity (Miller et al., 2009). Collectively, the data demonstrates that while Axins and Nkd1 proteins can physically interact, they function at different levels of β-catenin to antagonize Wnt signaling: Axins degrade β-catenin while Nkd1 prevents its nuclear accumulation.

The interaction between Axins and Nkd1 is curious given their unique modes of action and it is unclear how they would function together to inhibit Wnt signaling. Alternatively, it could be that the interaction is merely a mechanism to aggregate Wnt signaling components together to nucleate inhibitors (Colozza and Koo, 2021). Based on available information, we speculate that these two regulators function independently of one another to provide differential regulation of the pathway, not just on or off as we now know that different levels of β-catenin have different consequences on gene transcription and ultimately cell function (Albuquerque et al., 2002; Rieger et al., 2016). Thus, we hypothesized that compared with both single mutants, the combined loss of Axin2 and Nkd1 would result in a synergistic phenotype, resulting in hyperactivated or hypersensitized Wnt signaling. Counter to our hypothesis, we found that, for the most part, Axin2 and Nkd1 function in a linear manner, where Nkd1 functions downstream of Axin2 to antagonize Wnt/β-catenin signaling.

RESULTS

Knocking out Axin2 and Nkd1

The Axin2 and Nkd1 knockout lines were created by injecting sgRNA/Cas9 with their respective sgRNAs at the one cell stage and grown to adulthood (F0). These were back crossed into their respective genetic lines and F1 founders were tail clipped and genotyped via indel detection by amplicon analysis followed by sequencing. We evaluated several mutants for each gene, all of which showed similar phenotypes (unpublished data) and thus we only focused on one genotype for each of axin2 and nkd1. The axin2gh1/gh1 zebrafish were made in the tail long background (TL) whereas the nkd1gh2/gh2 zebrafish were generated in the Tubingen (TU) background. The axin2gh1/gh1 mutant has a 12-nucleotide deletion with a 1 nucleotide insertion in exon 2 leading to a frameshift mutation and premature stop codon p.Gly64Aspfs*65 (Supplemental Figure 1). The nkd1gh2/gh2 mutation is a 16-nucleotide deletion in exon 3 leading to a frameshift mutation and premature stop codon p.Asn60Glnfs*79 (Supplemental Figure 2). The axin2gh1/gh1;nkd1gh2/gh2 zebrafish were created by crossing axin2+/gh1 zebrafish with nkd1gh2/gh2 to create axin2+/gh1;nkd1+/gh2. The axin2+/gh1;nkd1+/gh2 zebrafish were then crossed to nkd1gh2/gh2 to create axin2+/gh1;nkd1gh2/gh2, in which axin2+/gh1;nkd1gh2/gh2 zebrafish were incrossed to make axin2gh1/gh1;nkd1gh2/gh2.

For reference, in the figure legends, capital “N” refers to the number of biological replicates, whereas lowercase “n” refers to the total number of observations. In addition, all mutants discussed below are maternal-zygotic mutants unless otherwise specified.

Wnt regulator mutants have developmental defects.

Our first objective was to determine whether the different mutants had developmental defects and to report on those that we consistently observe. While some of the experiments reported below have only one biological replicate, all have been observed multiple times and in different genetic backgrounds. We report only on the ones where we have included all the relevant controls.

The Wnt regulator mutants have no overt phenotype, appear phenotypically wild type until early adult hood (see below), and are viable. Upon close examination it was demonstrated that the mutants have heart looping defects (Figure 1A). Specifically, at 2 d postfertilization (dpf), wild type larvae had D looped hearts 90.6% of the time, whereas, the axin2gh1/gh1, nkd1gh2/gh2, and axin2gh1/gh1;nkd1gh2/gh2 had D looped hearts 41.3, 53.8, and 26.9% of the time, respectively. In each of the Wnt regulator mutants there was an increase in proportion of D incomplete looped, L looped, L incomplete looped, and unlooped hearts when compared with wild type. Because heart looping phenotypes can be variable based on the genetic background, we injected wild type embryos with sgRNA/Cas9 targeting both Axin2 and Nkd1 and observed a similar proportion of heart looping phenotypes compared with the knockout lines (Figure 1B). These results support research on Wnt signaling in heart development where apc mutant zebrafish also develop with unlooped hearts, although at a much greater proportion (Lin and Xu, 2009). Furthermore, past research has demonstrated that knock down of Nkd1 with morpholinos impairs heart development by inducing a heart jogging defect (Schneider et al., 2010).

FIGURE 1: Wnt regulator mutants have developmental defects. (A) 2 dpf Zebrafish embryos were observed for heart looping phenotypes and quantified in (B). (C) Zebrafish were treated with PTU from 1–5 dpf and then fixed at 5 dpf for neuromast staining with terminal neuromasts highlighted by the red box and quantified in (D). (E) Maternal-zygotic adult axin2gh1/gh1 zebrafish occasionally develop a curved spine phenotype. (F) Adults from a heterozygous incross were phenotyped for the curved spine followed by genotyping. (G) Maternal-zygotic adult axin2gh1/gh1 zebrafish often have a receding operculum phenotype. (H) Adults from a heterozygous incross were phenotyped for the receding operculum followed by genotyping. Error bars represent SEM, and a Chi-Square analysis was used with **** = p value < 0.0001.

We also examined other known Wnt dependent events for signs of activated Wnt signaling. Wnt signaling is necessary for neuromast formation and migration where it works alongside FGF signaling for proper patterning (Ma and Raible, 2009; Valdivia et al., 2011; Head et al., 2013). Hyperactivation of Wnt signaling has previously been shown to reduce neuromast migration in zebrafish which lead us to investigate whether neuromast migration is impaired in the Wnt regulator mutants (Figure 1C; Head et al., 2013). At 5dpf, wild type zebrafish had five to seven terminal neuromast, whereas axin2gh1/gh1, nkd1gh2/gh2, and axin2gh1/gh1;nkd1gh2/gh2 double mutants had four to six, four to seven, and zero to six terminal neuromast, respectively. To confirm that hyperactivation of Wnt causes reduced neuromast migration, wild type embryos were treated with a small molecule Wnt activator, 6-bromoindirubin-3′-oxime (BIO), which caused larvae to have zero to four terminal neuromasts at 5dpf (Figure 1D). Total number of neuromasts for all Wnt regulator mutants did not change (unpublished data) suggesting that the migration of the neuromasts are perturbed, consistent with published reports (Head et al., 2013).

We also noticed a curved spine phenotype in the axin2gh1/gh1 mutants. An incross of axin2+/gh1 mutants resulted in 42 adults, of which, 2/6 axin2gh1/gh1, 0/25 axin2+/gh1, and 0/11 axin2+/+ developed with a curved spine phenotype (Figure 1, E and F). An incross of axin2gh1/gh1 mutants produced a similar frequency of phenotypes (∼20–50% of homozygous mutants), suggesting that there is no maternal contribution. The axin2gh1/gh1;nkd1gh2/gh2 zebrafish also develop a curved spine, albeit with less frequency, whereas nkd1gh2/gh2 single mutants do not (unpublished data). This suggests that the genetic background (TL in the axin2gh1/gh1 mutants and predominantly TU in the axin2gh1/gh1;nkd1gh2/gh2 mutants) does not have a significant role. This phenotype is not surprising given Wnt signaling is important in bone formation where both hyperactivation and hypoactivation of the pathway leads to skeletal deformities (Yu et al., 2005; Hayes et al., 2014; Qu et al., 2021). Furthermore, Axin2 knockout mice have axial skeletal defects and a small head phenotype (Yu et al., 2005).

Further, the axin2gh1/gh1 adult zebrafish often develop with receding opercula (Figure 1, G and H). In contrast to the low to moderate penetrance of the curved spine, axin2gh1/gh1 mutants from an incross of axin2+/gh1 had complete penetrance (6/6) of receding opercula in the one cross that we performed whereas axin2+/gh1 and axin2+/+ had 9/25 and 2/11 with receding opercula, respectively, suggesting that there may be a dosage effect (Figure 1H). Operculum malformation is common in stressed or old age zebrafish, however, axin2gh1/gh1 mutants consistently develop with a receding operculum at an early age (∼5 mo). The axin2gh1/gh1;nkd1gh2/gh2 often have receding opercula at a frequency seen in the axin2gh1/gh1 mutants, whereas, nkd1gh2/gh2 mutants do not (unpublished data).

The varying degree in the number of terminal neuromasts suggests that the Wnt regulator mutant larva might have altered responses to stimulation. Therefore, we assessed 5dpf Wnt regulator mutants for swimming behavior and tap response. The larvae were recorded for 1.5 h under dark conditions using a 24-well plate and assayed for thigmotaxis. We discovered that the wild type larvae had a greater thigmotactic behavior when compared with the Wnt regulator mutants. Furthermore, while wild type larvae spent 82.6% of their time along the edge of the well, axin2gh1/gh1, nkd1gh2/gh2, and axin2gh1/gh1;nkd1gh2/gh2 mutants spent 70.6, 70.9, and 75.4%, of their time along the edge, respectively. Startle response was measured for 30 taps over a period of 30 s with the first three taps in the analysis showing the greatest difference in response when comparing the Wnt regulator mutants to wild type larvae. The axin2gh1/gh1 larvae had a mild reduction in startle response whereas the nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 larvae had a severe reduction in startle response when compared with wild type (Supplemental Figure 3).

Wnt regulator mutants have differential sensitivities to exogenous Wnt8.

While Axin2 and Nkd1 are both negative feedback regulators of Wnt signaling acting at the level of β-catenin, we have previously demonstrated that exogenous Nkd1 is only active in the presence of ligand activated Wnt signaling (Larraguibel et al., 2015). In contrast, Axin2 does not require the Wnt ligand for its activation (Larraguibel et al., 2015). Therefore, we wanted to assess the response of the regulator mutants to overactive Wnt signaling. Overexpression of wnt8 leads to an eyeless phenotype that is easily visible at 1dpf (Figure 2, A and B). Given that both Axin2 and Nkd1 are negative feedback regulators, we hypothesized that loss of these regulators would exacerbate the eyeless phenotype induced by Wnt8 overexpression. Injection of a low dose of wnt8 where only 4.7% of embryos are eyeless in wild type, increased to 33.8 and 31.6% in axin2gh1/gh1and nkd1gh2/gh2 mutants, respectively, supporting our hypothesis (Figure 2C). We further expected the axin2gh1/gh1;nkd1gh2/gh2 double mutants to be even more sensitive than either of the single mutants but surprisingly, only 4.6% of axin2gh1/gh1;nkd1gh2/gh2 larvae were eyeless, suggesting the loss of one regulator rescues the effect of the loss of the other (Figure 2C). Furthermore, nkd1gh2/gh2 injected mutants had a large proportion of embryos develop with a short-twisted axis when wnt8 was overexpressed, which was rarely seen in either axin2gh1/gh1 single or axin2gh1/gh1;nkd1gh2/gh2 double mutants (Figure 2, D–F). This suggests that loss of Axin2 can rescue loss of Nkd1 in the short- twisted axis phenotype, but it is unknown whether this is the case in the eyeless phenotype.

FIGURE 2: Wnt regulator mutants have differential sensitivities to exogenous Wnt8. (A–C) Overexpression of 100 pg of wnt8 in wild type embryos results in the classic Wnt gain of function eyeless phenotype in ∼ 5% of embryos, but more than 30% in axin2gh1/gh1 and nkd1gh2/gh2 mutants. Small eyes were considered to have eyes. In contrast, the axin2gh1/gh1;nkd1gh2/gh2 resembles the wild-type phenotype (C). (D–F) Analysis of body axis development revealed that only the nkd1gh2/gh2 mutants develop a short-twisted axis with exogenous wnt8 in about 30% of the embryos. (G) The Wnt reporter line was used to assess Wnt activity at the midbrain-hindbrain boundary at 1dpf when either Axin2 and/or Nkd1 was knocked down using sgRNA/Cas9. In each of the sgRNA/Cas9 injections there was an expansion of Wnt activity in the forebrain when compared with wild type embryos. Single plane brightfield images were overlaid with composite GFP images and as such the eye is not visible in all images. Error bars represent SEM, different letters above the bar signify significance with a p value < 0.01 using a one-way ANOVA.

To further explore the effect of knocking out Axin2 and/or Nkd1 on Wnt activity we utilized the Wnt reporter zebrafish line (7XTCF:GFP; Moro et al., 2012). We injected axin2 and/or nkd1 sgRNAs along with Cas9 protein into one-cell stage transgenic embryos derived from the Wnt reporter line (Crispants). Wnt activity via GFP expression in the brain was then analyzed at 1dpf. Uninjected wild type embryos predominantly have GFP expression at the midbrain-hindbrain boundary with some activity being present in the forebrain (Figure 2G). Knock down of Axin2 caused an anterior-ward expansion in GFP expression in the forebrain, whereas knock down of Nkd1 only caused a mild increase in GFP expression in the forebrain. When both Axin2 and Nkd1 were knocked down there was a similar expansion in GFP expression as seen in the axin2 crispants alone, suggesting the double crispant is more similar to the axin2 crispant but the variability in the data precludes statistical testing. To further explore this, we overexpressed wnt8, which caused axin2 crispants to have a major anterior expansion in GFP expression which was milder in the nkd1 or axin2;nkd1 crispant embryos (Figure 2G). Importantly, the eyeless phenotype induced in this transgenic line mimicked the knockouts where the single crispants had increased sensitivity while the double crispants showed the same insensitivity to exogenous Wnt8 (unpublished data).

Thus far, loss of either Axin2 or Nkd1 produces results that are consistent with their roles as negative feedback regulators; however, results become counterintuitive when both Axin2 and Nkd1 are simultaneously knocked out, especially in response to exogenous Wnt8. Therefore, we wanted to test how these mutants responded when Wnt signaling was overactivated in a ligand independent manner. To accomplish this, we used the small molecule BIO, which inhibits Gsk3β activity (Figure 3, A and B; Meijer et al., 2003). Wild type embryos treated with 0.5µM of BIO reduces the size of the eye approximately 45.7% (±19.9%; Figure 3C). Using the same dose of BIO, axin2gh1/gh1, nkd1gh2/gh2, and axin2gh1/gh1;nkd1gh2/gh2 double-mutant eyes were reduced by 69.5% (± 21.6%), 96.1% (± 3.9%), and 89.4% (± 0.42%), respectively (Figure 3C; Supplemental Figure 4). To ensure that the sensitivity to BIO was not a consequence of the different genetic backgrounds we created axin2gh1/gh1 mutants from an axin2+/gh1 incross. Here, axin2gh1/gh1 embryos treated with BIO had an increase in the eyeless phenotype when compared with embryos with at least one copy of wild type Axin2 from the same cross demonstrating that the sensitivity is specific for loss of Axin2 (Supplemental Figure 5). In stark contrast to the effect of ectopic Wnt8, the axin2gh1/gh1;nkd1gh2/gh2 double mutant was highly sensitive to BIO. This suggests that a ligand, or receptor proximal, dependent event is somehow inhibiting Wnt/β-catenin signaling in the absence of two intracellular negative feedback regulators during early gastrulation when canonical Wnt signaling is patterning the hindbrain (Moens and Prince, 2002).

FIGURE 3: axin2gh1/gh1;nkd1gh2/gh2 mutants are sensitive to activation at the destruction complex level. (A) Embryos were treated with varying concentrations of BIO from 4 – 30 hpf with representative images taken of the wild type embryos. (B) In wild type embryos, the area of the eye has a decreasing linear association with the concentration of BIO with all embryos being eyeless at 1.5 µM. In contrast, the majority of the axin2gh1/gh1;nkd1gh2/gh2 mutant embryos were eyeless at 0.5 µM of BIO. (C) The axin2gh1/gh1;nkd1gh2/gh2 mutant has no change in the size of eye in the control treatment but has significantly smaller eyes at 0.5 µM of BIO when compared with wild type revealing that the axin2gh1/gh1;nkd1gh2/gh2 mutants are sensitive to BIO. Measurements were performed on imageJ and a t test was used with ** = p value < 0.01. All error bars represent SEM. No statistics were performed on the axin2gh1/gh1 and nkd1gh2/gh2 single mutants.

Wnt regulator mutants have differential effects on Wnt target genes.

The data so far suggests that regulation of Wnt by negative feedback regulators is a complex process. To further explore the effect of Wnt8 and determine whether we could separate out how these two regulators function to restrict Wnt signaling, we analyzed the expression of several well characterized Wnt target genes. At 30 and 50% epiboly axin2 expression is restricted to the ventral lateral domain of the developing zebrafish embryo. Overexpression of Axin1 abolished axin2 expression at 30 and 50% epiboly, whereas hyperactivating Wnt signaling using a stable form of β-catenin (ΔN-β-catenin) causes ectopic axin2 expression (Supplemental Figure 6). Combined with published results, this confirms that Axin2, along with Nkd1 and Sp5a are excellent markers for active Wnt signaling during pregastrulation development (Weidinger et al., 2005; Dunty et al., 2014; Moya et al., 2014; Mukherjee et al., 2022).

We first used qRT-PCR to quantify the expression of the Wnt target genes axin2, nkd1, and sp5a at 30 and 50% epiboly (when zygotic Wnt signaling is high) with or without overexpression of wnt8 in the different genetic backgrounds (Figure 4; Jho et al., 2002; Van Raay et al., 2007; Huggins et al., 2017). We did not observe a decrease in endogenous axin2 or nkd1 expression in any of the Wnt regulator mutants, suggesting that the mutations in axin2 and nkd1 are not inducing nonsense mediated decay. In the axin2gh1/gh1 mutants, there was a predictable, albeit slight, increase in all three target genes that was exacerbated in the presence of exogenous Wnt8 at both 30 and 50% epiboly. Curiously, only the expression of axin2 in the axin2gh1/gh1 mutants was significant at 30% epiboly (Figure 4, A and B). Conversely, the nkd1gh2/gh2 mutants displayed a somewhat different pattern, as there was no substantive increase in either axin2 or nkd1 expression at 30% with or without exogenous Wnt8 when compared with controls (Figure 4C). At 50%, the expression of axin2 and nkd1 in nkd1gh2/gh2 was similar to the axin2gh1/gh1 mutant in that there was a slight, but non-significant increase in their expression that was moderately enhanced by exogenous Wnt8 (Figure 4D). Interestingly and curiously, sp5a expression in uninjected nkd1gh2/gh2 mutants was not significantly impacted but skyrocketed in the presence of exogeneous Wnt8 at 30% epiboly (Figure 4C). Furthermore, at 50% epiboly, uninjected nkd1gh2/gh2 mutants had a significant increase in sp5a expression that was further exacerbated by the addition of exogenous Wnt8 (Figure 4D). Importantly, the axin2gh1/gh1;nkd1gh2/gh2 mutant closely resemble the nkd1gh2/gh2 mutants with respect to all three genes (Figure 4E). This strongly suggests that each single mutant has a unique consequence on the activation of Wnt target genes and that they might be functioning at different levels of the pathway.

FIGURE 4: Wnt regulator mutants have differential effects on Wnt target genes. qRT-PCR was performed at 30% (A and C) and 50% (B, D, and E) epiboly for the Wnt target genes axin2, nkd1, and sp5a. Target gene expression was normalized to actin and made relative to WT uninjected expression levels. Injections were performed with 100 pg of wnt8 at the one cell stage. Expression of axin2, nkd1, and sp5a tended to increase in axin2gh1/gh1 mutants at both 30 and 50% epiboly but only axin2 expression increased significantly at 30% epiboly. However, in the nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 mutants, axin2 and nkd1 expression did not increase substantially compared with wild type. In contrast, sp5a expression showed a significant increase at 30 and 50% epiboly in the nkd1gh2/gh2 mutants and at 50% in the axin2gh1/gh1;nkd1gh2/gh2 (A–E, error bars represent SEM, * = p value < 0.05, ** = p value < 0.01, one-way ANOVA). Injection of Wnt8 into wild type (column 3) is often significant to uninjected wild type (column 1), but this is not shown for clarity.

Wnt regulator mutants have differential effects on gsc expression.

To explore this differential effect further, we chose to look at the expression of gsc by whole mount in situ hybridization (WMISH) to determine how the dorsal organizer is affected by loss of Wnt feedback regulation. The dorsal organizer is under the influence of both maternal and zygotic canonical Wnt signaling (Kelly et al., 2000; Bellipanni et al., 2006). From dome to 50% epiboly, the arc of gsc expression on the dorsal side of the embryo can be quantified as a read out for Wnt activity. This would also determine whether there was an expansion of Wnt signaling or ectopic Wnt signaling. At dome stage and 30% epiboly there was no significant difference in the arc of gsc expression in the axin2gh1/gh1 and nkd1gh2/gh2 maternal-zygotic mutants when compared with wild type (Figure 5, A and B). In contrast, at 50% epiboly, the axin2gh1/gh1 maternal-zygotic (TL background) embryos had an 8.1° increase in gsc arc angle which was not seen in either the nkd1gh2/gh2 maternal-zygotic (TU background) or axin2gh1/gh1;nkd1gh2/gh2 maternal-zygotic embryos (predominately TU background; Figure 5, C and D).

FIGURE 5: Wnt regulator mutants have differential effects on gsc expression. (A–D) WMISH for gsc was performed on embryos at three different stages for axin2gh1/gh1 and nkd1gh2/gh2 single mutants (A and B) as well as 50% epiboly for all Wnt regulator mutants (C) and measured for the arc of gsc expression (D). At 50% epiboly, the arc angle of gsc expression was significantly broader in the axin2gh1/gh1 embryos, which was not seen in either nkd1gh2/gh2 or axin2gh1/gh1;nkd1gh2/gh2 mutants. (E and F) Analysis of gsc expression on embryos at 50% epiboly injected with 100 pg of wnt8 from either an axin2+/gh1;nkd1+/gh2 incross (E) or an axin2+/gh1;nkd1gh2/gh2 incross (F). (E) The axin2gh1/gh1;nkd1± and axin2±;nkd1gh2/gh2 embryos had an increase in gsc arc angle when compared with axin2±;nkd1± . (F) In contrast, axin2gh1/gh1;nkd1gh2/gh2 double mutant embryos showed no increase in gsc arc angle when compared with the single knockout genotypes. Injections were performed at the one cell stage and arc angle measurements were taken on ImageJ. Embryos were genotyped using indel detection by amplicon analysis and PCR based methods. A one-way ANOVA was used for all statistics with * = p value < 0.05, error bars represent SEM (A, B, C, and E) and SD (F).

This was further validated by knocking down Axin2 using morpholinos which caused an increase in gsc arc angle at 50% epiboly but not at 30% epiboly (Supplemental Figure 7). This is in contrast to previous research from our lab where knocking down Nkd1 using a morpholino increased gsc expression at 30% epiboly (Van Raay et al., 2007). This may be a consequence of knocking down translation via morpholino versus knocking out maternal and zygotic transcription in the nkd1gh2/gh2 or axin2gh1/gh1;nkd1gh2/gh2 knockouts shown here. This suggests that Axin2, but not Nkd1, normally functions to restrict the expression of gsc.

To better evaluate whether the genetic background is a contributing factor (for example, the TL may be more sensitive than the TU background), we overexpressed wnt8 in embryos that were generated from a double heterozygote incross (equal parts TU and TL), processed for WMISH, scored for phenotype, and then genotyped. Unlike the axin2gh1/gh1;nkd1gh2/gh2 maternal-zygotic mutants above, these embryos would be zygotic mutants for all three genotypes with an approximately equal contribution of the TL and TU genetic backgrounds. Embryos with Axin2 knocked out (axin2gh1/gh1;nkd1±) had a 14.4° increase in gsc arc angle, whereas, embryos with Nkd1 knocked out (axin2±;nkd1gh2/gh2) had a 6.6° increase in gsc arc angle when compared with embryos with at least one wild type allele of both Axin2 and Nkd1 (axin2±;nkd1±), both of which were significant (Figure 5E). Similarly to the results in the axin2gh1/gh1 maternal-zygotic (TL background), the axin2gh1/gh1;nkd1± (TU/TL background) had the greatest increase in gsc arc angle with wnt8 overexpression and does not support the notion that the TL background is more sensitive.

The evaluation of axin2gh1/gh1;nkd1gh2/gh2 from the double heterozygote incross was hindered by the low numbers that were available, as only 1/16 of all offspring from a double heterozygous cross would be axin2gh1/gh1;nkd1gh2/gh2. Therefore, to increase the number of axin2gh1/gh1;nkd1gh2/gh2 embryos, wnt8 overexpression was performed on embryos from a axin2+/gh1;nkd1gh2/gh2 (predominantly TU background) incross. Twenty five percent of these embryos would be zygotic axin2gh1/gh1 and maternal-zygotic nkd1gh2/gh2. Similar to the maternal-zygotic mutants experiments above (Figure 5, A–D), wnt8 injected axin2gh1/gh1;nkd1gh2/gh2 mutants did not have an increase in gsc (Figure 5F). Collectively, this suggests that loss of Nkd1 rescues the loss of Axin2 or that Nkd1 functions downstream of Axin2 and further suggests that the effect of loss of Axin2 on gsc expression appears to be independent of genetic background and maternal sources.

The evidence thus far suggests that there are at least three different responses in the loss of the negative feedback regulators: 1) Additive: terminal neuromasts are more severe in axin2gh1/gh1;nkd1gh2/gh2 mutant; 2) Counteractive: axin2gh1/gh1;nkd1gh2/gh2 rescues Wnt8 overexpression in single mutants, and 3) Nkd1 downstream: sp5a expression is up in nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 mutants but not in axin2gh1/gh1; gsc and axin2 expression up in axin2gh1/gh1, but there is no change in nkd1gh2/gh2 or axin2gh1/gh1;nkd1gh2/gh2. Further, we only observed the counteractive response in the eyeless phenotype, suggesting that this may be related to other signaling phenomenon occurring during patterning of the eye field.

RNA-seq analysis suggests axin2 gh1/gh1;nkd1 gh2/gh2 double mutants are more similar to nkd1 gh2 /gh2.

To better understand the relationship between these two Wnt regulators, we performed RNA-sequence analysis on embryos at 50% epiboly, a time point when we observed significant effects on gene expression in the mutants. In addition to the overall analysis in gene expression, we also attempted to identify groups that reflect the three different responses identified above.

RNA-sequencing was performed on the Wnt regulator mutants as well as the two wild-type background genotypes: TU and TL. The two genetic backgrounds were useful in identifying and removing genetic variability unrelated to the mutations. Three biological replicates were isolated on different days, but from the same parents for each genotype. RNA-sequencing was performed using paired-end reads at a depth of 50 million reads.

A principal component analysis was performed on all genes. This revealed that all biological replicates were closely related to each other demonstrating the reproducibility of the dataset. Further, the axin2gh1/gh1 mutants were more similar to the TL background from which it was derived, while the nkd1gh2/gh2 mutants clustered closely with the axin2gh1/gh1;nkd1gh2/gh2 double mutants (Figure 6A), which is consistent with the predominantly TU background in the double mutants. Furthermore, a heat map of the top 1000 differential regulated genes also shows a similar trend where axin2gh1/gh1;nkd1gh2/gh2 embryos are more similar to nkd1gh2/gh2 (Figure 6B). This data fits very well with the sp5a expression we observed above. To further explore the data, Venn diagrams were created to identify overlap in differentially expressed genes (DEG) found in each of the Wnt regulator mutants. Before the Venn diagrams were created, genes that were differentially expressed (FDR < 0.05) between TU and TL were removed to strengthen the reliability of our data by removing genes that were variable between the background genotypes. Using an absolute log2FC >2 and FDR < 0.05 filtering method revealed 735 DEG in the axin2gh1/gh1 mutants, 869 DEG in nkd1gh2/gh2 mutants and 312 DEG in the axin2gh1/gh1;nkd1gh2/gh2 (Figure 6, C and D; Supplemental Table S6–S9). We attributed the reduced number of DEG in axin2gh1/gh1;nkd1gh2/gh2 mutants to the fact that they were filtered against both the TU and the TL backgrounds, while the single mutants were only filtered against their respective backgrounds.

FIGURE 6: axin2gh1/gh1;nkd1gh2/gh2 embryos have a more similar RNA profile to nkd1gh2/gh2 than to axin2gh1/gh1 . RNA-seq was performed at 50% epiboly for all Wnt regulator mutants as well as two wild type background genotypes: TU and TL. (A) PCA analysis and a (B) heat map analysis of the top 1000 differentially regulated genes shows that that axin2gh1/gh1;nkd1gh2/gh2 double mutant embryos have a similar RNA profile to the nkd1gh2/gh2 single mutant embryos. (C–D) Venn diagrams were made for upregulated genes (C) (log2FC > 2 and an FDR < 0.05) and down regulated genes (D) (log2FC < -2 and an FDR < 0.05) with genes that were significantly different (FDR < 0.05) between TL and the TU backgrounds being removed. (E) A gene list for the 34 genes that were upregulated and the 15 genes that were down regulated for each of the Wnt regulator mutants was made with the log fold change values provided for comparison. For more information on DEG for each Wnt regulator mutant refer to Supplemental Tables 6–9.

From this, we conclude that the clustering of the nkd1gh2/gh2 mutants with axin2gh1/gh1;nkd1gh2/gh2 and both of these away from the axin2gh1/gh1mutants is not due to the number of genes that are affected. Instead, it may be due to gene expression levels found in the mutants. Although not significant, a comparison of the expression values of the 34 upregulated genes shared in all mutant genotypes demonstrated that loss of Nkd1 had a greater impact on gene expression levels compared with loss of Axin2 (Supplemental Figure 8). A similar trend was observed in the 15 genes they had in common that are downregulated. Importantly, this was only observed for the shared genes, as comparison of expression levels of all the DEG for each genotype revealed nearly identical violin plots (Supplemental Figure 8C).

While both single mutants each have several hundred DEGs we were still perplexed by the lack of overlap in the number of shared genes. We wanted to determine whether this was a consequence of reduced expression levels of genes in the axin2gh1/gh1 that were not shared. That is, do the 735 genes (outside of the 34 shared ones) found to be differentially expressed in axin2gh1/gh1 have differential expression in the nkd1gh2/gh2 mutant but just don’t reach the threshold of significance? To test this, we evaluated the raw expression levels of the 735 DEG identified in the axin2gh1/gh1 mutants in the nkd1gh2/gh2 dataset. For comparison, we also performed the reciprocal analysis (Supplemental Figure 9). This analysis demonstrates that the nonoverlapping DEG in the axin2gh1/gh1 remain relatively unchanged in the nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 double mutants (Supplemental Figure 9, A and B). This suggests that modifications to the transduction of the Wnt signal in the axin2gh1/gh1 mutant are very different to the modifications that occur with the loss of Nkd1 and supports the notion that these antagonists are functioning at different levels of the pathway.

Of the 34 common genes that were upregulated and 15 common genes that were downregulated in all the Wnt regulator mutant genotypes, a large portion (28) of the 49 DEG were uncharacterized with the vast majority of these (11/15) being downregulated (Figure 6E).

To determine whether the RNA-seq data supports the “additive” response group, we identified genes that were up (or down) in both axin2gh1/gh1 and nkd1gh2/gh2 mutants by Log2FC > 1 whose expression was exacerbated up (or down) in axin2gh1/gh1;nkd1gh2/gh2 mutants. This identified one gene, Prkag2b, that had a 5.5 fold change in both axin2gh1/gh1 and nkd1gh2/gh2 mutants and a 7.0 and 7.2-fold increase in the axin2gh1/gh1;nkd1gh2/gh2 versus TU and versus TL, respectively. There were eight genes (Supplemental Table 1) that were additive in their reduced expression.

To determine whether there was evidence for the “counteractive” response, we identified genes that were up (or down) by Log2FC > 1 in both the single mutants (same direction) but were either unchanged or down (or up) by less than onefold in the axin2gh1/gh1;nkd1gh2/gh2 mutants. This identified seven genes (Supplemental Table 2), all of which had unchanged expression in the axin2gh1/gh1;nkd1gh2/gh2 mutants.

To determine how many genes were in the “Nkd1 downstream” group, we selected genes that were differentially expressed by an absolute Log2FC > 1 in both the nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 mutants, but were unchanged in the axin2gh1/gh1 mutants. This identified 237 genes. Increasing the stringency to absolute Log2FC > 2 resulted in 74 genes and a further increase to absolute Log2FC > 3 resulted in 33 genes (Supplemental Table 3). Thus, while there is no significant support for “additive” or “counteractive” responses in the RNA-seq data, there is clearly support for the Nkd1 downstream response. We also determined whether there was an “Axin2 downstream” group that was differentially expressed in axin2gh1/gh1 and axin2gh1/gh1;nkd1gh2/gh2 by an absolute Log2FC > 1 , but were unchanged in the nkd1gh2/gh2 mutant and identified only five genes (Supplemental Table 4).

Analysis of the 237 “Nkd1 downstream” genes by GO and Panther analysis revealed no significant hits, regardless of the stringency we used. Curiously, only three of the 237 genes were related to Wnt signaling: gsk3b, fzd10 and caveolin. We performed similar GO analyses on each of the three mutant genotypes and in each case, there were no significant hits. Analysis of the nonsignificant hits did not reveal any patterns; however, broad neuronal systems were abundant. Surprisingly, Wnt signaling did not appear in any of the GO analyses which could be due to the analysis focusing on the proteins in the pathway rather than the expression of Wnt target genes (unpublished data).

Despite the high levels of sp5a identified in our qRT-PCR analysis, this gene was not identified as being a significant hit in the RNA-seq data. Thus, we wanted to investigate this gene and other known Wnt target genes. Using the RNA-seq data, a list was created of 59 known Wnt target genes identified by the Wnt signaling community. It should be noted that many of these are not unique to Wnt signaling and may have a more significant role in other pathways. Of the 59 Wnt target genes, 15 of them had differential expression in axin2gh1/gh1;nkd1gh2/gh2 mutant embryos (FDR < 0.05 vs. TU and FDR < 0.05 vs. TL). Of these 15 DEG, nine of them had similar expression to sp5a seen earlier by qRT-PCR: fsta, lef1, nkd1, six3b, sox17, sox2, sp5a, tbx3a, and tiam1a (Supplemental Figure 10). The inclusion of sp5a in the list is validation of the qRT-PCR data. Furthermore, the levels of axin2 and nkd1 in the RNA-seq data are also consistent with our qRT-PCR analysis, showing modest but consistent increases in expression in all three mutant genotypes.

Collectively, the differential gene expression analysis suggests that loss of Nkd1 has a greater impact on gene expression levels compared with the loss of Axin2 and that there is no additive, synergistic or counteractive effect in the loss of both Nkd1 and Axin2. While the GO analysis did not reveal any significant hits, there was a modest, but consistent effect on Wnt target genes. Further, it seems that there are a significant number of uncharacterized genes that are targets of the Wnt pathway that have yet to be described, highlighting the complexity and depth of this pathway in multipotent progenitor cells.

The lack of overlap in target genes between axin2gh1/gh1 and nkd1gh2/gh2 (only 73 genes out of a combined 1531) is curious but does support our qRT-PCR and gsc expression analysis. This suggests that each contribute to modifications of β-catenin-mediated signaling resulting in an altered transcriptional response, which may include alternative transcriptional start sites in their shared genes. To explore this further, we evaluated differential transcript usage (DTU) in the different genetic backgrounds but found no obvious pattern for DTU when comparing the Wnt regulator mutants. Several of the most significant hits are shown in Supplemental Figure 11. Accordingly, we did not pursue this further.

Knocking out Axin2, but not Nkd1, increases cytoplasmic levels of β-catenin.

As both Axin2 and Nkd1 interact with β-catenin in the cytoplasm, we next wanted to determine whether there were changes in the levels of cytoplasmic and nuclear β-catenin. As Axin2 is predicted to target β-catenin for ubiquitin mediated degradation, we predicted that loss of Axin2 would result in more cytoplasmic β-catenin. In contrast, we have previously demonstrated that Nkd1 functions to inhibit the nuclear accumulation of β-catenin and so its loss is predicted to result in more nuclear β-catenin but not necessarily a change in cytoplasmic levels (Van Raay et al., 2011). To test this, we first performed Western blot analysis on cytoplasmic fractions from 30% epiboly embryos, a time point where we have previously shown cytoplasmic β-catenin levels to be sensitive to Wnt signaling hyperactivation (Van Raay et al., 2011). Further, Pan-cadherin was used as a control to ensure that the cytoplasmic fractions were free of plasma membrane as β-catenin is localized to the membrane due to its interactions with cadherins (Tian et al., 2011). Compared to uninjected controls, wild type embryos had a relative cytoplasmic β-catenin increase of 1.86 when overexpressed with wnt8, which is consistent with our previous reports (Figure 7A; Van Raay et al., 2011). Analysis of β-catenin levels in the Wnt regulator mutants demonstrated a significant increase in axin2gh1/gh1mutants. In particular, axin2gh1/gh1 and axin2gh1/gh1;nkd1gh2/gh2 had a relative cytoplasmic β-catenin increase of 2.90 and 3.52, respectively, compared with their uninjected genotype control (Figure 7B). These were significantly higher than the 1.86 increase seen in wild type wnt8 injected embryos. By comparison, the nkd1gh2/gh2 mutant had a relative cytoplasmic β-catenin increase of 2.17, which was not significant when compared with wild type (Figure 7B) but supports the higher levels seen in the axin2gh1/gh1;nkd1gh2/gh2 mutant. This supports the model that Axin2 affects cytoplasmic levels of β-catenin (Zhang et al., 2007; Van Raay et al., 2011; Bernkopf et al., 2015). While the statistics support our model that loss of Nkd1 does not alter cytoplasmic β-catenin levels, we are not convinced that there is no change in nkd1gh2/gh2 mutants, especially when axin2gh1/gh1;nkd1gh2/gh2 shows a modest additive effect.

FIGURE 7: Knocking out Axin2, but not Nkd1, increases cytoplasmic levels of β-catenin. (A) Whole cell lysate and cytoplasmic fractions of embryos from wild type and Wnt regulator mutants, with or without 200 pg of wnt8 injected, were collected at 30% epiboly for western blot analysis for cytoplasmic levels of β-catenin. Pan-cadherin was used to confirm purity of the cytoplasmic fraction and Actin was used as a loading control. (B) Because each genotype was run on separate western blots, the increase in cytoplasmic β-catenin when wnt8 was overexpressed was made relative to their uninjected counterpart. Both axin2gh1/gh1 and axin2gh1/gh1;nkd1gh2/gh2 embryos had a greater increase in cytoplasmic β-catenin levels with wnt8 overexpression when compared with wild type embryos. Error bars represent SEM, * = p value < 0.05, one-way ANOVA.

With the increase in cytoplasmic β-catenin in at least the axin2gh1/gh1 mutants, we next wanted to evaluate the levels of β-catenin in the nucleus. In our experience, nuclear fractions cannot be sufficiently purified from β-catenin-containing membrane fractions in zebrafish embryos for western blot analysis, therefore we chose to perform immunohistochemistry at 30% epiboly using anti-β-catenin antibodies (Van Raay et al., 2007, 2011). We first evaluated nuclear β-catenin levels in the different genetic backgrounds along the ventrolateral domain which has endogenous Wnt signaling and did not observe any obvious difference in nuclear β-catenin levels (Supplemental Figure 12). We next set out to evaluate nuclear β-catenin in the presence of exogenous Wnt8. To do this, we injected a cocktail of Axin2 and/or Nkd1 sgRNAs, Cas9, and wnt8 along with a tracer molecule (Dextran or mcherry) into 1 blastomere of an eight-cell stage wild type embryo to generate mosaic crispants. Using this design, labeled cells would be Wnt8 and crispant positive, but juxtaposed cells would be crispant negative, but still receive the Wnt ligand (Van Raay et al., 2011). Knocking down Axin2 and/or Nkd1 did not have any significant difference in nuclear β-catenin levels when compared with uninjected juxtaposed cells (Supplemental Figure 13). It is possible that immunohistochemistry is not sensitive enough to detect subtle differences in nuclear β-catenin considering we are knocking down regulators of the pathway and not essential components.

Taken together, loss of Wnt regulation via Axin2 and Nkd1 results in an increase in cytoplasmic β-catenin (at least significantly for Axin2), but this does not translate into a measurable increase in nuclear β-catenin.

Wnt regulator mutants have lower expression of metabolism proteins.

The differences that we have observed in phenotypes, gene expression and cytoplasmic β-catenin levels suggests that Axin2 and Nkd1 have both unique and overlapping ways of controlling Wnt signaling and that this might be reflected in the protein complement in each of the mutants. We first tried to purify β-catenin to identify proteins that might be differentially interacting with it under the different genetic conditions; however, we were unable to purify sufficient levels of β-catenin from zebrafish embryos to perform this analysis. Instead, we decided to evaluate the entire protein pool using mass spectrometry with 30% blastocysts that have been rigorously purified away from yolk proteins. Using this strategy, we identified differentially expressed proteins between the Wnt regulator mutants. These results were variable between biological replicates which, despite our best efforts, was likely due to yolk contamination during isolation. Therefore, we chose samples with similar ionic chromatograms to perform our analysis (Figure 8A). From this, we discovered 53 differentially expressed proteins (FDR < 0.1, logFC ≥ 1, peptides ≥ 2) in the Wnt regulator mutants with the majority of the proteins being either ribosomal subunits or related to metabolism (Figure 8B). Furthermore, and similar to the RNA-seq data, we see that the axin2gh1/gh1;nkd1gh2/gh2 mutant is more similar to the nkd1gh2/gh2 mutant, whereas the axin2gh1/gh1mutant is similar to wild type; however, the proteins that were differentially expressed in mass spectrometry did not always correlate to differential expression in the RNA-seq results. Furthermore, in the nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 mutants, there is an overall trend of decreased protein expression levels. Of the 53 proteins, there were 18 proteins (Fabp3, Aldh2.2, Prdx1, ldhba, Pcxa, Tcp1, Igf2bp3, Pkma, Actb2, Cct5, Prdx6, Tpi1a, Prdx2, Ppa1b, Sod1, Cox17, Ranbp1, Stmn1a) that have a connection to Wnt signaling (Figure 8C). Interestingly, most of the proteins related to Wnt have been demonstrated to stimulate Wnt signaling, as knocking out/down the proteins leads to either reduced β-catenin levels or Wnt target gene expression. Finally, using string analysis, we identified the top KEGG pathways to be ribosomal proteins and pyruvate metabolism related proteins (Figure 8, D and E). While this technique requires refinement to generate high quality data it does support our model where Axin2 and Nkd1 have both overlapping and unique effects in regulating Wnt signaling.

FIGURE 8: Wnt signaling mutants have lower expression of metabolism proteins. Mass spectrometry was performed on embryos at 30% epiboly. (A) Only samples with similar total ionic chromatograms were used, resulting in only one biological replicate for each genotype. (B) Heat map of 53 differentially expressed proteins using PEAKS 10 software (FDR < 0.1, logFC ≥ 1, peptides ≥ 2). (C) Eighteen proteins were identified to have a connection to Wnt signaling in the literature: Fabp3 (Liu et al., 2013), Aldh2.2 (Zhao et al., 2015), Prdx1 (Zheng et al., 2018), Idhba (Mazzio et al., 2020), Pcxa (Lee et al., 2012), Tcp1 (Tang et al., 2020), Igf2bp3 (Song et al., 2023), Pkma (Lu et al., 2021), Actb2 (Gu et al., 2021), Cct5 (Li et al., 2022b), Prdx6 (Xu et al., 2019), Tpi1a (Xia et al., 2023), Prdx2 (Feng et al., 2020), Ppa1b (Niu et al., 2017), Sod1 (Chandrasekharan et al., 2021), Cox17 (Li et al., 2022a), Ranbp1 (Liu et al., 2019), Stmn1a (Zhang et al., 2019). (D and E) String protein networks were created using all proteins identified (D) and a network of the 39 nonribosomal proteins (E).

DISCUSSION

Our initial hypothesis was that Axin2 and Nkd1 uniquely and independently affect Wnt/β-catenin signaling with Axin2 regulating the stability of cytoplasmic β-catenin, while Nkd1 prevents its nuclear accumulation (Van Raay et al., 2011; Bernkopf et al., 2015). We further anticipated a lack of a significant phenotype in each of the single mutants, based on the phenotypes in their mammalian counterparts (Li et al., 2005; Yu et al., 2005; Zhang et al., 2007; Hulin et al., 2017). Nonetheless, we speculated that with the different modes of action, there would be a convergence on the transcriptional targets and we anticipated a synergistic effect in the axin2gh1/gh1;nkd1gh2/gh2 double mutants resulting in a more severe phenotype. Surprisingly, the phenotypic data was underwhelming. The heart looping data showed no evidence of epistasis or synergy. The axial and operculum defect in the axin2gh1/gh1 single and axin2gh1/gh1;nkd1gh2/gh2 double mutants suggests that Axin2 functions downstream of Nkd1, however, a detailed analysis clearly demonstrates that the axin2gh1/gh1;nkd1gh2/gh2 mutants resemble the ndk1gh2/gh2 mutants. This strongly suggests that Nkd1 functions downstream of Axin2.

Evaluation of the entire transcriptome in a whole organism is a double edge sword. On one hand, we are evaluating the loss of genes in their native environment, which is very powerful. On the other hand, not every cell is actively engaged in Wnt signaling which can generate significant background. To maximize the former and minimize the later, we chose to evaluate the transcriptional readout of the mutants at 50% epiboly as there is substantial zygotic Wnt signaling occurring throughout the ventrolateral domain of the blastula. The transcriptional data supports Axin2 having a separate mode of Wnt antagonism from Nkd1; however, we do not have any evidence that Axin2 functions downstream of Nkd1. In contrast, the majority of our analysis supports the model that Nkd1 functions downstream of Axin2 (Table 1).

TABLE 1: Summary of results. Each experiment is listed with the effects shown for each genotype. A (–) symbol signifies no observed effect, while the (+) symbol signifies an effect. + = somewhat affected, ++ = affected, +++ = very affected. Colour coding reflects genotype. In the majority of our analysis the axin2gh1/gh1;nkd1gh2/gh2 mutant looks similar to nkd1gh2/gh2 but rarely has a more severe phenotype when compared with the single mutants.

	

The difficulty in working with negative feedback regulators is that they function to regulate the intensity, spatial distribution and/or duration of the signal and can act as a safety mechanism more than an integral component of the pathway, much like an airbag in a car (Freeman, 2000). For example, we see a more robust response in sp5a expression when we ectopically express Wnt8 in the mutants compared with wildtype. Nonetheless, we still observed significant increases in target gene transcription without the addition of exogenous Wnt8. We also speculate that the high number of DEG’s identified is an overestimation of the actual targets. Increasing the absolute Log2FC to >3 resulted in 33 genes that are significantly differentially transcribed in both the nkd1gh2/gh2 single and axin2gh1/gh1;nkd1gh2/gh2 double mutants but remained unchanged in the axin2gh1/gh1 mutant. One, Gsk3βb, is linked to Wnt signaling and is upregulated over 60-fold; two are related to transcriptional activity; one is a histone deacetylase and nine are uncharacterized proteins with the remainder having various unrelated functions. Similarly for axin2gh1/gh1mutants with an absolute Log2FC > 1 but remained unchanged in the nkd1gh2/gh2or axin2gh1/gh1;nkd1gh2/gh2 mutants resulted in 52 genes, one of which is related to Wnt signaling (sec12I8); nine are related to DNA binding; four are related to histone function; 10 are uncharacterized proteins and the remainder have various unrelated functions (Supplemental Table 5). The significant hits in DNA binding and histone modifications are in line with the model that loss of Axin2 and/or Nkd1 would alter Wnt signaling transcriptional activity, but this requires further investigation.

While the data is consistent with Nkd1 functioning downstream of Axin2, there are a few scenarios where we observe unexpected phenotypes in the presence of ectopic Wnt8. First, Wnt8, but not BIO, induces a kinked tail phenotype in the nkd1gh2/gh2 mutants, which is rescued in axin2gh1/gh1;nkd1gh2/gh2 mutants. We speculate that the kinked tail phenotype is a result of perturbed Wnt/planar cell polarity (PCP) signaling, but how this is rescued by loss of Axin2 is unknown.

Second, Axin2 and Nkd1 are both expressed along the ventrolateral margin of the gastrulating embryo when the hindbrain is being patterned (Moens and Prince, 2002; Van Raay et al., 2007; Gao et al., 2016). Predictably, the single mutants were highly sensitive to exogenous Wnt8, resulting in the classic Wnt gain of function eyeless phenotype. Our prediction was that the axin2gh1/gh1;nkd1gh2/gh2 double mutant embryos would be even more sensitive, but in fact the opposite was true; the axin2gh1/gh1;nkd1gh2/gh2 mutant was insensitive to exogenous Wnt8, resulting in a wild-type eye phenotype. To explain this, we must consider the role of Nkd1 in antagonizing the noncanonical Wnt/PCP pathway. We have previously demonstrated that Nkd1 functions to inhibit noncanonical Wnt/PCP signaling (Van Raay et al., 2007). Furthermore, evidence suggests that there is cross talk between canonical Wnt and Wnt/PCP signaling where activation of Wnt/PCP can inhibit canonical Wnt/β-catenin signaling, however, this mechanism is not fully understood (Torres et al., 1996; Nemeth et al., 2007; Bisson et al., 2015; Bell et al., 2022). In this scenario, the presence of excess Wnt8 in combination with the loss of Nkd1 antagonism of Wnt/PCP during early gastrulation may lead to increased Wnt/PCP signaling and thus a higher inhibition of canonical Wnt signaling that could overcome the loss of Axin2 resulting in a rescue of the eyeless phenotype. In support of this model is the Wnt/PCP phenotype (kinked axis) that is induced only when Wnt8, and not BIO, is overexpressed in the nkd1gh2/gh2 mutants. Rescue of the eyeless phenotype through the loss of both Axin2 and Nkd1 suggests that the Wnt/PCP-Wnt/β-catenin interaction is occurring within the cytoplasm upstream of the destruction complex and is dependent on a Wnt ligand interacting with its receptor. As Dvls are involved in both Wnt/β-catenin and Wnt/PCP signaling (Sharma et al., 2018), bind Nkd1 and Axin1 but not Axin2 (Miller et al., 2009), act upstream of the destruction complex and are activated by Wnt ligands (Ma et al., 2020), Dvls seem to be likely candidates to participate in this phenomenon but this requires further investigation.

Consistent with our results, there have been several studies investigating gene expression under different Wnt signaling scenarios, and Sp5 expression appears to be a highly upregulated Wnt target gene in these studies (Dunty et al., 2014; Moya et al., 2014; Huggins et al., 2017; Mukherjee et al., 2022). In addition, sp5 expression is significantly higher than axin2 and nkd1 in these studies, which is also consistent with our study (Dunty et al., 2014; Moya et al., 2014; Huggins et al., 2017; Mukherjee et al., 2022). Similarly, when we compared our hits to the Wnt signaling study from Moya et al, 2014, we both identified cyb5d1, wdr78, gnao1b, and crb2a as Wnt target genes (Moya et al., 2014). Further, we saw both upregulation and downregulation of Wnt target genes which is consistent with the research by Ewing et al. (2018) where Wnt target genes were also upregulated and downregulated when comparing HCT116 Δ45-β-catenin to HCT116 wild type cells (Ewing et al., 2018). This is also consistent with the function of β-catenin-dependent transcription being context dependent which is covered in depth in the review by Valenta et al, (2012).

Our evaluation of the proteome by mass spectrometry is a unique approach as these studies are rarely performed on whole organisms, especially in zebrafish due to the high yolk protein content. While some replicates provided more hits, the poor reproducibility between replicates made it insufficient for any meaningful analysis and thus, we decided to focus on the samples with lower abundant proteins but with the most similar ion chromatogram profiles. This method identified 53 differentially expressed proteins with 18 of those proteins having known connections to Wnt signaling. For example, Prdx1 and Prdx2, proteins have been previously linked to enhancing β-catenin stability and were found to be downregulated in nkd1gh2/gh2 and axin2gh1/gh1;nkd1gh2/gh2 mutants in our results (Ewing et al., 2018; Zheng et al., 2018; Feng et al., 2020). The transcript levels of prdx1 were also significantly reduced, while prdx2 transcripts were only slightly reduced. However, the majority of the proteins we identified were related to ribosomal assembly or metabolism, which may reflect the high translation rates occurring at 50% epiboly, a time when the embryo has fully transitioned into zygotic expression (Vastenhouw et al., 2019). Interestingly, most of the proteins connected to Wnt signaling seem to have a promoting role in the pathway and are down regulated in the Wnt regulator mutants. This could suggest that the embryos are modulating metabolism to downregulate Wnt signaling when Axin2 and/or Nkd1 is/are knocked out. Thus, our results are consistent with Wnt signaling having a known role in metabolism at both the gene and protein levels, however this is far from comprehensive and will need to be investigated further (Sethi and Vidal-Puig, 2010).

The differences between loss of Nkd1 and loss of Axin2 might suggest that Axin2 and/or Nkd1 are not negative feedback regulators of Wnt, but the overlapping results strongly suggests that they are which is consistent with the literature (Van Raay et al., 2011; Bernkopf et al., 2015). In particular, their sensitivity to Wnt8 overexpression or BIO treatment are well established indicators of excess Wnt signaling (Hu et al., 2010; Robertson et al., 2014; Marsden et al., 2018). Furthermore, their common phenotypes in heart looping and neuromast migration and their effect on many Wnt target genes are also strong indicators that Axin2 and Nkd1 are involved in Wnt signaling.

Alternatively, it might be argued that the mutations in axin2gh1/gh1 and nkd1gh2/gh2 are not complete nulls, which would require reading through of the premature stop codons (Li and Zhang, 2019). In support of this, we do not observe nonsense mediated RNA decay in the qRT-PCR data. Without specific antibodies to Axin2 and Nkd1, it is difficult to confirm a complete null, but there is evidence to suggest they are both nulls. First, phenotypes that are common to both have similar effects; for example, we observe similar effects in heart looping and sensitivity to exogenous Wnt8 for both mutants. We also observe a significant increase in axin2 and sp5a expression in axin2gh1/gh1 and nkd1gh2/gh2 mutants, respectively, with and without exogenous Wnt8. These results were also validated using RNA-seq where we saw differential expression of Wnt target genes in the Wnt regulator mutants. Furthermore, the axin2gh1/gh1 mutant has a similar number of DEG compared with nkd1gh2/gh2. Finally, we have previously generated axin2 and nkd1 crispants using at least two unique, nonoverlapping sgRNAs and evaluated their phenotypes and target gene expression with or without Wnt8. All Crispants had similar eyeless phenotypes and axin2 and nkd1 expression patterns as observed here (Pasula and Guelph, 2016). Thus, while we cannot be 100% certain that we have complete nulls, we are confident that at the very least the majority of the Axin2 and Nkd1 proteins are nonfunctional. We also injected Nkd2 sgRNA/Cas9 into the nkd1gh2/gh2 mutants and found no evidence of compensation by Nkd2 (unpublished data).

In conclusion, we have provided evidence that Nkd1 functions downstream of Axin2 and the destruction complex, as loss of Axin2 has minimal effect on Wnt signaling when Nkd1 is knocked out. Further, the evidence clearly demonstrates that Axin2 and Nkd1 uniquely modify Wnt signaling which we speculate happens at the level of β-catenin. How these modifications affect gene transcription will be an active area of investigation and provide important insights into the complex regulation of a signaling pathway involved in many development processes and diseases.

MATERIALS AND METHODS

Request a protocol through Bio-protocol.

Axin2 and Nkd1 Gene deletions

The website chop chop (https://chopchop.cbu.uib.no/ ) was used to design sgRNAs for Axin2 and Nkd1, with sgRNAs being selected based on stringency and location (Labun et al., 2019). sgRNAs were made using the GeneArt Precision gRNA Synthesis Kit following the protocol provided in the kit (Invitrogen A29377, Supplemental Table 10). The sgRNA were mixed with InvitrogenTM TrueCut Cas9 protein (Thermo Fisher Scientific) and injected at the one-cell stage. Cas9 efficiency was determined by indel detection by amplicon analysis.

Neuromast staining

Embryos were treated with 0.003% PTU from 1–5 dpf to inhibit pigmentation. At 5 dpf larvae were fixed in 4% paraformaldehyde overnight at 4°C and then washed three times with phosphate-buffered saline with 0.1% Tween 20 (PBST). Embryos were washed three times in alkaline tris buffer (0.5M Tris pH 9.5, 50 mM MgCl2, 0.1M NaCl, 0.1% tween) and stained in alkaline tris buffer with 550 nM Nitro blue tetrazolium chloride (NBT, Roche), and 400 nM 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP, Roche).

Microinjections

Injections were performed using a PV820 pneumatic PicoPump microinjector (World Precision Instruments) with needles pulled using a Model P-1000 Flaming Brown Micropipette Puller (World Precision Instruments, TW100F-4). Needles were cut to yield an injection volume of ∼1 nL.

BIO

Embryos were arranged into a six-well dish at 4 hpf containing either 0 µM, 0.25 µM, 0.5 µM, 1 µM, and 1.5 µM of BIO with 1% DMSO. Embryos were incubated at 28.5°C until 30 hpf and were fixed using 4% PFA overnight at 4°C. Images were taken and measurements for the area of the eye were done using ImageJ. The Wnt regulator mutants were treated in parallel with WT embryos. For the BIO treatment on Axin2 mixed embryos, an incross of axin2+/gh1 zebrafish was performed and embryos were treated with 0.75µM of BIO with 1% DMSO. Phenotypes for the eyes were recorded at 1dpf and each embryo was genotyped using fragment analysis.

qRT-PCR

RNA was isolated from a pool of 10 embryos using The GENEzol TriRNA Pure Kit (FroggaBio). RNA was then directly used in the Luna Universal One-Step RT-qPCR Kit (New England BioLabs). Primers listed in Supplemental Table 10.

WMISH

Embryos were fixed at dome, 30, and 50% epiboly using 4% paraformaldehyde overnight at 4°C. Embryos were washed three times in PBST and WMISH was carried out following the protocol provided in the Thisse and Thisse manuscript (Thisse and Thisse, 2008).

RNA sequencing preparation and analysis

RNA was isolated from a pool of 10 embryos at 50% epiboly using the GENEzol TriRNA Pure Kit (FroggaBio). RNA samples were DNase treated using the Invitrogen DNA-free DNA Removal Kit (Thermo Fisher Scientific) and an RNA integrity number of more than 8.0 was confirmed for all samples using the 4200 Tapestation system (Agilent). Poly(A) mRNA library preparation was performed using NEBNext Ultra II DNA library prep kit for illumina (New England BioLabs) and 2 × 100bp paired-end sequencing was performed at a depth of 50 million reads on an Illumina Novaseq 6000 platform by the University of Toronto Donelly Sequencing Centre. The reads were aligned using Rsubread v2.2.6 (Liao et al., 2019) to the Ensembl Genome Browser assembly ID: GRCz11. EdgeR v3.30.3 (Robinson et al., 2010) was used to filter reads and DESeq2 v1.28.1 (Love et al., 2014) was used to perform differential expression analysis.

Western blotting

At 30% epiboly 10 embryos were manually deyolked using forceps and lysed using a 27-gauge needle in a TKM buffer (50 mM Tris-HCl pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.02% sodium azide, 1X SIGMAFAST protease inhibitor cocktail tablets EDTA free). An aliquot was taken for whole cell lysate and the rest of the sample was centrifuged at 100,000 × G for 1 h at 4°C. The supernatant containing the cytoplasm was collected, and the pelleted plasma membrane proteins was resuspended in TKM buffer. Westerns were performed using 1:1500 anti-β-catenin (BD Biosciences Catalogue# 610154), 1:2000 anti-Pan-cadherin (Abcam Catalogue# ab16505), and 1:5000 anti-Actin (Sigma Catalogue# A5441).

Mass spectrometry

Thirty embryos at 30% epiboly were dechorionated and placed in a deyolking buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3). Embryos were vortexed on the lowest speed for 5 min to dissolve the yolk and centrifuged at 300 × G to pellet cells. The supernatant was removed, and the cells were washed four times using a wash buffer (110 mM NaCl, 3.5 mM KCl, 10 mM Tris-HCl pH 8.0) followed by 300 g spin to pellet cells. Cells were then washed using 1X wash buffer with added EDTA free SIGMAFAST protease inhibitor cocktail tablets. Cells were centrifuged at 300 × G and the final supernatant removed. 500 µL of lysis buffer (8 mM Urea, 75 mM NaCl, 2 mM MgCl2, 10mL 1M Tris-HCl pH 8.2, 1X SIGMAFAST protease inhibitor cocktail tablets EDTA free) was added to the cells and sonicated for 10 min. Lysates were incubated at 4°C for 30 min and centrifuged at 13,000 × G. The supernatant was moved to a clean tube and the pellet was discarded. Twenty five microliters of 200 mM of iodoacetamide was added to the tubes and incubated for 30 min at room temperature in the dark. One milliliter of cold acetone was added, vortexed, and stored at -80°C to precipitate the protein. Protein pellets were then decanted, air dried, and resuspended in 50 mM of ammonium bicarbonate. Samples were digested using trypsin and incubated at 37°C overnight and dried using vacuum centrifugation. To concentrate the samples, C18 columns were used, and protein samples were loaded into the Thermo East nLC Orbitrap Exploris 240. Analysis was performed using PEAKS 10 software. Mass spectrometry was performed by the Advanced Analysis Centre at the University of Guelph.

Availability of data and materials

Data generated or analyzed during this study are included in this published article [and its supplementary information files]. Complete datasets generated for RNA sequencing during and/or analyzed during the current study are available in the NCBI Gene Expression Omnibus, accession: GSE246858. To review GEO accession GSE246858: Go to www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE246858.

Supplementary Material

We would like to thank Matt Cornish and Mike Davies at the Hagen Aqualab facility at the University of Guelph for supporting the maintenance of the zebrafish system. We would also like to thank Amber Park and Dyanne Brewer at the Advanced Analysis Centre at the University of Guelph for their support in mass spectrometry. Lastly, we would like to thank all current and previous members in the Van Raay lab for all their support.

Abbreviations used:

APC adenomatous polyposis coli

BIO 6-bromoindirubin-3’-oxime

DEG differentially expressed genes

DPF days post fertilization

DTU differential transcript usage

DVL dishevelled

GO gene ontology

GSC goosecoid

GSK3β glycogen synthase kinase-3β

HPF hours post fertilization

KEGG kyoto encyclopedia of genes and genomes

PBST phosphate-buffered saline with 0.1% TritonX-100

PCP planar cell polarity

PCR polymerase chain reaction

PFA paraformaldehyde

PTU 1-phenyl 2-thiourea

qRT-PCR quantitative reverse transcription polymerase chain reaction

sgRNA single guide ribonucleic acid

TL tail long

TU tübingen

WMISH whole mount in situ hybridization

WT wild type

Reviewer Report

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-02-0059-T) on April 24, 2024.
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